PROJECT SUMMARY By internalizing cell surface receptors, clathrin-mediated endocytosis enables cellular responses to external cues, regulates nutrient availability, and controls cell signaling. Clathrin-coated pits (CCPs) form at the plasma membrane through the coordinated assembly of dozens of adaptor and coat proteins into a mesh-like network that surrounds a budding vesicle. Within seconds of initiation, nascent CCPs will either mature productively into vesicles or abortively disassemble. What differentiates productive CCPs from their abortive counterparts? It has been suggested that productive CCPs pass through a critical checkpoint, but the checkpoint criteria remain poorly understood. Toward explaining the mechanism that drives CCP progression, preliminary work shows that key CCP initiator proteins, Fcho1 and Eps15, assemble together at membrane surfaces into liquid droplets. The formation of protein droplets via phase separation has recently been shown to provide spatiotemporal control over the catalysis of several pathways including membrane receptor signaling and actin filament nucleation. In a similar way, assembly of a protein droplet at the CCP could function to locally catalyze endocytosis. Fcho1 and Eps15, which are among the earliest factors to arrive at CCPs, recruit other clathrin adaptors and are collectively essential for timely CCP initiation and maturation. Importantly, these initiator proteins bear two hallmarks of phase separating proteins: multivalent interaction motifs and intrinsically disordered regions. Recently I have discovered that Fcho1 and Eps15 assemble into protein liquid droplets at membrane surfaces. This exciting result has the potential to explain both the stochastic assembly of nascent CCPs at discrete endocytic sites and the robust recruitment of a protein network to these sites. Specifically, phase separation of initiator proteins could provide a plausible mechanistic explanation for CCP initiation and maturation. Therefore, the goal of the proposed work is to understand the mechanism by which assembly of the Fcho1/Eps15 initiator proteins contributes to robust endocytosis. Work in Aim 1 will evaluate the biochemical properties of the Fcho1/Eps15 network, testing the working hypothesis that Fcho1 and Eps15 enhance recruitment of each other to the membrane through specific multivalent interactions, and thereby enhance recruitment of downstream binding partners. Work in Aim 2 will evaluate the biophysical properties of the Fcho1/Eps15 network, testing the working hypothesis that the thermodynamic and kinetic properties of Fcho1/Eps15 droplets are consistent with a phase separated system. Work in Aim 3 will evaluate the properties of the Fcho1/Eps15 network in live cells, testing the working hypothesis that the liquid-like behavior of Fcho1/Eps15 is essential for effective catalysis of CCP maturation in cells. The outcome of this research will be a characterization of the presently unknown thermodynamics and kinetics of the CCP initiator complex. More broadly, this work has the potential to introduce a new biophysical paradigm for understanding the roles of protein networks during vesicle formation events throughout the cell.